The Journal of Immunology, 2002, 169: 4153-4160.
Copyright © 2002 by The American Association of Immunologists
Enforced Expression of Bcl-2 Restores the Number of NK Cells, But Does Not Rescue the Impaired Development of NKT Cells or Intraepithelial Lymphocytes, in IL-2/IL-15 Receptor
-Chain-Deficient Mice1
Masahiro Minagawa*,
Hisami Watanabe
,
Chikako Miyaji
,
Katsuhiro Tomiyama*,
Hideki Shimura*,
Akiko Ito*,
Masaaki Ito*,
Jos Domen2,
,
Irving L. Weissman
and
Kazuhiro Kawai3,*
Departments of
* Dermatology and
Immunology, Niigata University School of Medicine, Niigata, Japan; and
Departments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305
 |
Abstract
|
|---|
IL-2/IL-15R
-deficient mice display impaired development of NK
cells, NKT cells, and intraepithelial lymphocytes of the intestine and
skin. To determine the role of survival signals mediated by IL-2/IL-15R
in the development of these innate lymphocytes, we introduced a
bcl-2 transgene into IL-2/IL-15R
-deficient mice.
Enforced expression of Bcl-2 restored the number of NK cells in
IL-2/IL-15R
-deficient mice, but the rescued NK cells showed no
cytotoxic activity. The numbers of NKT cells and intestinal
intraepithelial lymphocytes did not increase significantly, and skin
intraepithelial lymphocytes remained undetectable in the
bcl-2 transgenic IL-2/IL-15R
-deficient mice. These
results indicate an essential role of IL-2/IL-15R-mediated survival
signals in the development of NK cells, but they also show that
additional nonsurvival signals from IL-2/IL-15R are necessary for
innate lymphocyte development.
 |
Introduction
|
|---|
Mutations
in the common cytokine receptor
-chain
(
c),4
which is a shared subunit of the receptors for IL-2, IL-4, IL-7, IL-9,
IL-15, and IL-21 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11), result in X-linked SCID in
humans (12). Similarly, mice with targeted mutations in
the
c gene exhibit impaired development of
multiple lymphocyte lineages characterized by reduced numbers of
conventional 
T cells and B cells and absence of 
T cells,
NK cells, and intraepithelial lymphocytes (IEL) of the intestine and
skin (13, 14, 15, 16). Analyses of mice deficient for an
individual
c-dependent cytokine or cytokine
receptor subunit revealed IL-7/IL-7R as crucial for the development of
several major lymphocyte lineages. Thus, mice deficient for IL-7 or
specific
-chain of IL-7R (IL-7-/- and
IL-7R
-/- mice) have reduced numbers of
conventional 
T cells and B cells and no 
T cells
(17, 18, 19, 20, 21, 22). However, NK cells and TCR
intestinal IEL
develop almost normally in these mice (18, 21, 22, 23). In
contrast, mice deficient for the shared
-chain of IL-2R and IL-15R
(IL-2/IL-15R
-/- mice) display normal
development of conventional 
T cells and B cells but have
dramatically reduced numbers of NK cells, TCR
and
TCR
CD8
subsets of intestinal IEL, and
NK1.1+TCR
+ NKT cells,
and no skin IEL (23, 24, 25, 26, 27). Because IFN regulatory
factor-1-/- mice (in which the IL-15
gene expression is impaired (28, 29)),
IL-15-/- mice, and
IL-15R
-/- mice are similarly deficient in NK
cells, NKT cells, and CD8
intestinal IEL (28, 29, 30, 31),
and because IL-15-/- mice have no skin IEL
(32), the development of these innate lymphocytes may
depend primarily on IL-15/IL-15R rather than IL-2/IL-2R. In fact,
IL-2-/- mice do not display obvious defects in
the development of NK cells, NKT cells, and skin IEL (26, 33, 34), although impaired development of CD8
intestinal IEL
in IL-2-/- mice has been demonstrated
(23, 34, 35).
Multiple signaling pathways from IL-7R and IL-2/IL-15R have been
identified (36), but the signals directly regulating the
lymphocyte development are not fully defined. The protein tyrosine
kinases Janus kinase (Jak)1, which is associated with IL-7R
and
IL-2/IL-15R
(7, 8, 37), and
c-associated Jak3 (7, 8, 38) are
critical to invoke signals from IL-7R and IL-2/IL-15R (39, 40). Accordingly, both Jak1-/- and
Jak3-/- mice display defects in the lymphocyte
development similar to those of
c-deficient
mice (39, 41, 42, 43, 44). Essential roles of two distinct
downstream signals in the lymphocyte development have been also
identified for the IL-7R (45). An important IL-7R-mediated
signal is to promote V(D)J recombination in the IgH and
TCR
gene loci in B cell and 
T cell
precursors, respectively (46, 47, 48, 49). Although a role of
IL-7R-mediated signals in TCR
locus rearrangements has been also
suggested (50), the thymic development of conventional

T cell precursors is blocked before TCR
rearrangement in
IL-7-/-, IL-7R
-/-,
c-deficient, and
Jak3-/- mice (17, 20, 23, 44, 51).
The thymic precursors in these mice exhibit reduced expression of the
antiapoptotic protein Bcl-2 (44, 52, 53, 54), and enforced
expression of Bcl-2 by introducing a bcl-2 transgene
restores the development of conventional 
T cells, but not B
cells or 
T cells in IL-7R
-/- and
c-deficient mice (51, 53, 55, 56, 57, 58). Therefore, the primary role of IL-7R in the
conventional 
T cell development is to provide survival
signals in their thymic precursors independently of the signals
promoting VDJ recombination of the TCR
locus.
Comparably little is known about the downstream signals from the
IL-2/IL-15R that regulate innate lymphocyte development. Unlike IL-7,
IL-2/IL-15 does not activate signals promoting V(D)J recombination in
lymphocyte precursors. However, multiple signaling pathways from the
IL-2/IL-15R lead to the expression of antiapoptotic proteins Bcl-2 and
Bcl-xL in various cell lines as well as in
primary T cells (59, 60, 61, 62, 63, 64, 65). In addition, IL-2/IL-15 has been
shown to promote survival of mature human NK cells (66, 67) and mouse intestinal TCR
IEL (68) through
up-regulation of Bcl-2 expression. Therefore, the impaired development
of innate lymphocytes in the mice with abrogated IL-2/IL-15R-mediated
signals might result from the lack of survival signals.
To determine the role of survival signals mediated by IL-2/IL-15R in
the innate lymphocyte development, we have introduced a
bcl-2 transgene into
IL-2/IL-15R
-/- mice. We show that enforced
expression of Bcl-2 in IL-2/IL-15R
-/- mice
has differential effects on the development of each innate lymphocyte
lineage.
 |
Materials and Methods
|
|---|
Mice
IL-2/IL-15R
-/- mice and
H2K-bcl-2 transgenic mice were reported previously
(24, 69) and were maintained on a C57BL/6 background. The
bcl-2-IL-2/IL-15R
-/-
mice were generated by crossing
IL-2/IL-15R
-/- mice with
H2K-bcl-2 transgenic mice. C.B-17 SCID mice were
purchased from Japan Clea (Tokyo, Japan). All mice were used at 45 wk
of age.
Cell preparation
Liver lymphocytes, intestinal IEL, and epidermal cells were
prepared as described previously (27, 70, 71).
Flow cytometry
Cells were resuspended in PBS supplemented with 2% FCS, 0.1%
NaN3, and 25 mM EDTA. After preincubation with
anti-Fc
II/IIIR mAb (clone 2.4G2; BD PharMingen, San Diego, CA),
cells were stained with saturating amounts of the following mAbs (BD
PharMingen): FITC- or biotin-conjugated anti-TCR
(H57-597), PE-
or biotin-conjugated anti-NK1.1 (PK136), PE-conjugated DX5,
PE-conjugated anti-CD3-
(145-2C11), FITC-conjugated
anti-Mac-1 (M1/70), FITC-conjugated anti-Ly-49A (A1),
FITC-conjugated anti-Ly-49C/I (5E6), FITC-conjugated anti-Ly-49D
(4E5), FITC-conjugated anti-Ly-49G2 (4D11), PE-conjugated
anti-TCR
(GL3), FITC-conjugated anti-CD8
(53-6.7),
PE-conjugated anti-CD8
(53-5.8), and PE-conjugated anti-TCR
V
3 (536). Biotin-conjugated mAb was visualized with
streptavidin-TRI-color (Caltag Laboratories, Burlingame, CA) or
streptavidin-Quantum Red (Sigma-Aldrich, St. Louis, MO). After gating
on forward and side scatter and propidium iodide, viable cells were
analyzed using the FACScan flow cytometer with the Lysis II or
CellQuest program (BD Biosciences, Mountain View, CA). Intracellular
staining of transgenic or endogenous Bcl-2, IL-4, or IFN-
was
performed as described (53, 72) using the Cytofix/Cytoperm
kit (BD PharMingen) and the following mAbs: FITC-conjugated
anti-human Bcl-2 (126; DAKO, Glostrup, Denmark), FITC-conjugated
anti-mouse Bcl-2 (3F11), PE-conjugated anti-IL-4 (11B11),
PE-conjugated anti-IFN-
(XMG1.2), and PE-conjugated rat IgG
isotype control (R3-34) (all from BD PharMingen).
Cytotoxicity assay
Mice were injected i.p. with 100 µg of poly(I):poly(C)
(Pharmacia Biotech, Piscataway, NJ) in 100 µl PBS on days 0 and 1. On
day 2, effector cells were prepared and cytotoxic activity against
NK-sensitive YAC-1 target cells was determined by the standard 4-h
51Cr release assay (73).
Cytokine production assay
To induce IFN-
production by NK cells in vitro, spleen cells
were cultured (1 x 106 cells/ml) for 4
h in the presence or absence of 1 ng/ml mouse rIL-12 (PeproTech,
London, U.K.) and 100 ng/ml mouse rIL-18 (MBL, Nagoya, Japan).
GolgiStop (BD PharMingen) containing monensin was added during the last
2 h, and intracellular accumulation of IFN-
in NK cells was
analyzed by flow cytometry.
To stimulate NKT cells in vivo, mice were injected i.p. with 10 µg of
-galactosylceramide (
GalCer) (KRN7000; Kirin Brewery, Tokyo,
Japan). After 90 min, spleen cells were prepared and cultured (5
x 106 cells/ml) for 1 h. IL-4 levels of the
supernatants were determined using an ELISA kit (Genzyme/Techne,
Minneapolis, MN). For intracellular staining, cells were incubated in
the presence of GolgiStop for 2 h, and intracellular accumulation
of IL-4 was analyzed by flow cytometry.
RT-PCR
RNA extraction and reverse transcription using
pd(N)6 primers were performed as described
(27). Diluted cDNA was amplified by PCR using primers
specific for TCR V
14-J
281 (74). The amount of
template cDNA was normalized using primers for
-actin
(27).
Immunofluorescence staining
Five-micrometer frozen sections were cut and fixed in cold
acetone. The sections were stained with anti-MHC class II mAb
(clone M5/114) or rat IgG isotype control (BD PharMingen) and
visualized with FITC-conjugated anti-rat Ig Ab (DAKO).
Statistical analysis
Differences in the absolute numbers of each lymphocyte population
were analyzed for statistical significance using the Student
t test.
 |
Results
|
|---|
Generation of bcl-2-IL-2/IL-15R
-/- mice
H2K-bcl-2 transgenic mice, in which the human
bcl-2 transgene is driven by the H2Kb
promoter and Moloney murine leukemia virus long terminal repeat
(69), express the transgenic Bcl-2 in all hematopoietic
cells including thymic precursors of conventional 
T cells, B
cell precursors and hematopoietic stem cells in the bone marrow, and
spleen NK cells (51, 69). We also confirmed expression of
the transgenic Bcl-2 by NK cells and NKT cells in the liver, all
intestinal IEL subsets, skin IEL, and their fetal thymic precursors in
H2K-bcl-2 transgenic mice (data not shown).
IL-2/IL-15R
-/- mice display impaired
development of NK cells, NKT cells, and IEL of the intestine and skin
but have normal numbers of conventional 
T cells and B cells
(23, 24, 25, 26, 27). However, in aged
IL-2/IL-15R
-/- mice, conventional 
T
cells are spontaneously activated and induce an exhaustive
differentiation of B cells into plasma cells, resulting in fatal
lymphoproliferative disorders characterized by lymphadenopathy,
splenomegaly, high serum concentrations of autoantibodies, anemia, and
marked infiltrative granulocytopoiesis (24). These
immunopathological phenotypes were not altered in the aged
bcl-2-IL-2/IL-15R
-/- mice (data
not shown). Therefore, in the following experiments,
bcl-2-IL-2/IL-15R
-/- and
littermate control mice were used at 45 wk of age to minimize
possible influences of the lymphoproliferative disorders on the innate
immune cell development.
Enforced expression of Bcl-2 restores the number, but not cytotoxic
activity, of NK cells in IL-2/IL-15R
-/- mice
Enforced expression of Bcl-2 in
IL-2/IL-15R
-/- mice resulted in a
significant increase in the number of
NK1.1+TCR
- NK cells in
the liver and spleen (Figs. 1
and 2
), which was comparable to wild-type
(+/+) levels. The relative proportion of
NK1.1-TCR
+ conventional
T cells was decreased by the enforced expression of Bcl-2 (Fig. 1
),
probably due to the increase in the number of B cells. The
NK1.1+TCR
- cells in
bcl-2-IL-2/IL-15R
-/- mice were
CD3-
-TCR
-, and
Mac-1, DX5, Ly-49A, C/I, D, and G2 were expressed at similar levels by
NK cells in the spleens of +/+,
IL-2/IL-15R
-/-, and
bcl-2-IL-2/IL-15R
-/- mice (Fig. 3
and data not shown), Therefore, the
rescued NK cells in
bcl-2-IL-2/IL-15R
-/- mice were
phenotypically indistinguishable from mature NK cells in +/+ mice.
However, the spleen or liver lymphocytes in
bcl-2-IL-2/IL-15R
-/- mice showed
no cytotoxic activity against NK-sensitive target cells (Fig. 4
and data not shown), although the
proportion of NK cells in the effector cells of
bcl-2-IL-2/IL-15R
-/- mice
(6.3 ± 1.1% in the liver lymphocytes) was comparable to that of
+/+ mice (5.3 ± 1.7%).

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FIGURE 2. Restoration of NK cell number in
bcl-2-IL-2/IL-15R -/- mice. Absolute
numbers of NK cells in the liver and spleen of the mice with indicated
genotypes were calculated from the total cell number and the percentage
of NK cells. The mean and SD of three mice are shown. *, Significant
decreases in numbers of NK cells as compared with those in +/+ mice
(p < 0.01).
|
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It is known that NK cells produce large amounts of IFN-
in response
to stimulation with IL-12 and IL-18 (75). Because
cytotoxic activity and IFN-
production are regulated by different
pathways in NK cells (76), we next examined the capacity
to produce IFN-
of the rescued NK cells in
bcl-2-IL-2/IL-15R
-/- mice. Upon
in vitro stimulation with IL-12 and IL-18, spleen NK cells from +/+ and
bcl-2-IL-2/IL-15R
-/- mice rapidly
produced IFN-
(Fig. 5
). Thus, the
rescued NK cells in
bcl-2-IL-2/IL-15R
-/- mice have no
cytotoxic activity but can produce IFN-
.
Enforced expression of Bcl-2 does not restore the number of NKT
cells in IL-2/IL-15R
-/- mice
NKT cells are defined as T cells expressing the common NK cell
marker NK1.1 and skewed TCR composed of invariant V
14-J
281 chain
paired preferentially with polyclonal V
8.2 chain, which recognize
CD1d-bound glycolipid ligands (77). NKT cells secrete
immunoregulatory cytokines including IL-4 rapidly upon activation
(77). In contrast to NK cells, the number of
NK1.1+TCR
+ NKT cells in
the liver and thymus did not increase significantly in
bcl-2-IL-2/IL-15R
-/- mice (Figs. 1
and 6
). Because a small number of NKT
cells were present in IL-2/IL-15R
-/- and
bcl-2-IL-2/IL-15R
-/- mice (Figs. 1
and 6
), and V
14-J
281 mRNA could be detected in both mice (data
not shown), we examined the function of residual NKT cells in these
mice. As shown in Fig. 7
, NKT cells in
IL-2/IL-15R
-/- and
bcl-2-IL-2/IL-15R
-/- mice could
generate a rapid, though diminished, IL-4 response after in vivo
stimulation with their specific ligand
GalCer (78).
Fewer NKT cells were present in the spleen cells prepared from
IL-2/IL-15R
-/- mice injected with
GalCer
than in those from +/+ mice, but the proportion of IL-4-producing cells
in the NKT cell population of IL-2/IL-15R
-/-
mice was comparable to that of +/+ mice (Fig. 8
). In addition, NKT cells were the
primary population that produced IL-4 after in vivo stimulation with
GalCer in both +/+ and IL-2/IL-15R
-/-
mice (data not shown). Therefore, the diminished IL-4 production in
IL-2/IL-15R
-/- and
bcl-2-IL-2/IL-15R
-/- mice in
response to
GalCer stimulation in vivo would be attributed mainly to
the reduced number of NKT cells in these mice.
Enforced expression of Bcl-2 does not restore the number of
intestinal IEL in IL-2/IL-15R
-/- mice
Intestinal IEL are divided into several subsets based on the
expression of TCR
/
and CD4/CD8
/CD8
(79).
IL-2/IL-15R
-/- mice display selective
reduction in the numbers of the TCR
and TCR
CD8
subsets of intestinal IEL (23, 25), which are believed to
develop extrathymically (79). In
bcl-2-IL-2/IL-15R
-/- mice, the
relative proportion of the TCR
and TCR
CD8
subsets of
intestinal IEL appeared to increase slightly as compared with those in
IL-2/IL-15R
-/- mice (Fig. 9
). However, the increases in absolute
numbers of these subsets were not statistically significant (Fig. 10
), because of the bcl-2
transgene-induced reduction of the total number of intestinal IEL (Fig. 9
), which was due to decreases of the TCR
and TCR
CD8
subsets (Figs. 9
and 10
). The mechanism of the reduction of intestinal
IEL number in bcl-2-IL-2/IL-15R
+/+
mice is currently unknown. Interestingly, MHC class II expression by
villus intestinal epithelial cells, which is regulated by the TCR
subset of intestinal IEL (80), was not down-regulated in
IL-2/IL-15R
-/- mice (Fig. 11
).
Enforced expression of Bcl-2 does not rescue the development of
skin IEL in IL-2/IL-15R
-/- mice
Skin IEL in normal mice originate from early fetal thymocytes and
express an invariant TCR composed of V
3 and V
1 chains
(81). In IL-2/IL-15R
-/- mice,
fetal thymic V
3+ precursors develop almost
normally, but V
3+ cells are completely absent
in the adult skin (27). As shown in Fig. 12
, V
3+ skin
IEL remained undetectable in adult
bcl-2-IL-2/IL-15R
-/-
mice.
 |
Discussion
|
|---|
The importance of IL-15/IL-15R in the NK cell development has been
documented in previous studies (25, 28, 29, 30, 31, 82, 83, 84, 85). In
this study, we show that enforced expression of Bcl-2 restores the
number of NK cells in IL-2/IL-15R
-/- mice.
Because endogenous Bcl-2 expression by the residual NK cells in
IL-2/IL-15R
-/- mice is not significantly
diminished (our unpublished data), the increase in the number of NK
cells in bcl-2-IL-2/IL-15R
-/-
mice would not result from the accumulation of "leaky" NK cells
through Bcl-2-mediated prolonged survival. Therefore, an important role
of IL-15R in the NK cell development may be to provide survival signals
in their precursors at certain critical developmental stages. Although
cytokines other than IL-2/IL-15 also induce nonredundant survival
signals in NK cell precursors in vitro (84), these
cytokines and IL-15 may be involved in different stages of the NK cell
development (84, 86).
Because the
c is shared by other cytokine
receptors, IL-15 may activate specific signals promoting the survival
of NK cell precursors through IL-2/IL-15R
. Discrete domains of the
IL-2/IL-15R
are known to be involved in inducing expression of the
antiapoptotic proteins Bcl-2 and Bcl-xL
(59, 60, 61, 62, 63, 64, 65). The cytoplasmic tail of the IL-2/IL-15R
chain
is divided into three functional domains (87). The
membrane-proximal domain containing the serine-rich region (S-region)
recruits Jak1 and is essential to invoke downstream signals (8, 88, 89). The intermediate domain containing the acidic region
(A-region) interacts with Src family protein tyrosine kinases and an
adapter molecule Shc, and activates Ras/mitogen-activated protein
kinase and phosphatidylinositide 3-kinase/Akt signaling pathways
(36, 62, 90). The C-terminal half region (H-region) is
essential for the recruitment and activation of Stat5 (90, 91). Analysis of the IL-2/IL-15R
-/-
mice reconstituted with a mutant form of IL-2/IL-15R
lacking either
the cytoplasmic A- or H-region has revealed the importance of the
H-region in the NK cell development (92). In contrast, the
A-region is dispensable for NK cell development (92),
although A-region-dependent activation of Ras/mitogen-activated protein
kinase and phosphatidylinositide 3-kinase/Akt signaling pathways also
induces Bcl-2 and Bcl-xL expression (59, 61, 62, 63, 64). Therefore, among multiple signaling pathways from
IL-2/IL-15R
, which lead to induction of Bcl-2 and
Bcl-xL, the H-region-dependent Stat5 pathway
(60, 65) may provide nonredundant signals for the survival
of NK cell precursors. A crucial role for the Stat5 signaling pathway
in NK cell development is further supported by the absence of NK cells
in Stat5-/- mice (93, 94).
It was reported that enforced expression of Bcl-2 cannot rescue the NK
cell development in
c-deficient mice
(51). Therefore,
c-dependent
cytokine receptors other than IL-2/IL-15R may also play a role in the
NK cell development by providing critical signals distinct from
survival signals. Previous in vitro studies indicate that these signals
could be mediated by IL-7R (86, 95), although
IL-7-/- and IL-7R
-/-
mice display normal NK cell development (18, 21, 22). It
is possible that the IL-7R-mediated signals required for NK cell
development might be compensated for by additional signals from
IL-2/IL-15R in vivo. Alternatively, a
c-dependent cytokine receptor other than IL-7R
or IL-2/IL-15R might be involved in NK cell development. Although newly
identified IL-21R is a likely candidate for such a receptor (10, 11, 96), IL-21R-/- mice have recently
been shown to display normal NK cell development (97).
Interestingly, the rescued NK cells in
bcl-2-IL-2/IL-15R
-/- mice do not
have cytotoxic activity. Similar developmental arrest of NK cells at a
noncytotoxic state and restoration of their cytotoxic activity with
exogenous IL-15 have been demonstrated in the bone marrow-ablated mice
(83). Therefore, IL-15R-mediated signals are crucial not
only for the survival of NK cell precursors but also for the
acquisition of cytotoxic activity by NK cells. Because noncytotoxic NK
cells in the bone marrow-ablated mice express relatively low levels of
Ly-49 receptors (83), IL-15R-mediated signals might be
required for the expression of activating Ly-49 receptors by NK cells
to recognize target cells. However, we have confirmed that comparable
levels of inhibitory and activating Ly-49 receptors are expressed by
the noncytotoxic NK cells in IL-2/IL-15R
-/-
and bcl-2-IL-2/IL-15R
-/- mice and
by the cytotoxic NK cells in +/+ mice. Alternatively, induction of
cytotoxic proteins in NK cells might depend on IL-15R-mediated signals.
The latter possibility is supported by the observation that perforin
expression is regulated by IL-2/IL-15R
-mediated Stat5 activation
(98).
In contrast, the rescued NK cells in
bcl-2-IL-2/IL-15R
-/- mice can
produce IFN-
. In a recent study (99), in vivo
developmental stages of NK cells have been defined, and expansion and
functional maturation (acquisition of cytotoxic activity and the
capacity to produce IFN-
) of
NK1.1+Ly-49+DX5+Mac-1low
immature NK cells was shown to be accompanied by the up-regulation of
Mac-1 expression. Most residual spleen NK cells in
IL-2/IL-15R
-/- mice express Mac-1 at high
levels, and these phenotypically "mature" NK cells increase
significantly by enforced expression of Bcl-2. The "mature" NK
cells in bcl-2-IL-2/IL-15R
-/-
mice have the capacity to produce IFN-
, but not cytotoxic activity.
Therefore, up-regulation of Mac-1 expression, expansion, and
acquisition of the capacity to produce IFN-
and cytotoxic activity,
which occur during the final maturation steps in NK cell development,
may be regulated by different signaling pathways. In addition,
proliferation of NK cell precursors at this developmental stage may not
be mediated directly by signals from IL-15R.
NKT cells and the TCR
and TCR
CD8
subsets of
intestinal IEL are reduced, but not absent, in
IL-2/IL-15R
-/- mice (23, 25, 26). IL-2/IL-15R-mediated signals do not appear to be crucial
for the functional maturation of the precursors of these cells, because
at least some functions of residual NKT cells and TCR
intestinal
IEL in IL-2/IL-15R
-/- mice are not
compromised. Therefore, the precursors of NKT cells and intestinal IEL
would be able to differentiate to some extent without IL-2/IL-15R, and
IL-2/IL-15R-mediated signals might be important for their survival
and/or expansion. Because enforced expression of Bcl-2 does not restore
the numbers of NKT cells or intestinal IEL in
IL-2/IL-15R
-/- mice, the primary role of
IL-2/IL-15R in the development of these cells may be to provide
proliferation signals.
The absence of V
3+ skin IEL in adult
IL-2/IL-15R
-/- mice is primarily due to the
impaired survival and/or expansion of V
3+
cells in the fetal skin, rather than the developmental block of their
fetal thymic precursors (27). Somewhat surprisingly, in
light of these previous observations, enforced expression of Bcl-2
cannot rescue skin IEL in adult
IL-2/IL-15R
-/- mice. However, it should be
noted that some forms of apoptotic cell death, including that mediated
by the Fas-Fas ligand interaction, cannot be prevented by Bcl-2
(100). Therefore, IL-2/IL-15R
might provoke survival
signals in skin IEL independently of the induction of Bcl-2-related
antiapoptotic proteins. Alternatively, continuous expansion of skin IEL
through IL-2/IL-15R-mediated proliferation signals might be required
for their maintenance in the skin. Our results are consistent with
recent observations that introduction of the bcl-2 transgene
fails to rescue skin IEL in V
3 TCR-transgenic
IL-2/IL-15R
-/- mice (101).
In conclusion, this study has revealed an important role for the
survival signals from IL-2/IL-15R, which are mediated through the
induction of Bcl-2-related antiapoptotic proteins, in the development
of NK cells. However, additional signals from IL-2/IL-15R are required
for the acquisition of cytotoxic activity by NK cells, expansion of the
precursors of NKT cells and intestinal IEL, and maintenance of skin
IEL. As was the case for the two distinct signals from IL-7R regulating
the development of major lymphocyte lineages, heterogeneous signals
from IL-2/IL-15R may play differential roles in the development of each
innate lymphocyte lineage.
 |
Acknowledgments
|
|---|
We thank K. Ikuta, K. Akashi, H.
Suzuki, T. W. Mak, and H. Kawamura for providing
mice and mAbs and for helpful discussions, T. Ohteki for critically
reviewing the manuscript, T. Imai for technical assistance,
and Kirin Brewery for
GalCer (KRN7000).
 |
Footnotes
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|---|
1 This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (to K.K.). 
2 Current address: Department of Medicine, Division of Medical Oncology and Transplantation, Duke University Medical Center, Durham, NC 27710. 
3 Address correspondence and reprint requests to Dr. Kazuhiro Kawai, Department of Dermatology, Niigata University School of Medicine, 1-757 Asahimachi-dori, Niigata 951-8510, Japan. E-mail address: kawai{at}med.niigata-u.ac.jp 
4 Abbreviations used in this paper:
c, common cytokine receptor
-chain;
GalCer,
-galactosylceramide; IEL, intraepithelial lymphocyte; Jak, Janus kinase. 
Received for publication February 5, 2002.
Accepted for publication August 6, 2002.
 |
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